As noted in our project on the Role of Selenium in Cancer and Health, selenium is an essential micronutrient in the diet of humans and it has many health benefits. Furthermore, as also noted in the Role of Selenium in Cancer and Health project, we proposed that the health benefits are due in large part to the presence of selenium in selenoproteins as the selenium-containing amino acid, selenocysteine (Sec); and therefore, our program focuses 1) on the means by which Sec is biosynthesized and incorporated into protein and 2) on developing mouse models to assess the role of selenium and specific selenoproteins in human health and cancer prevention. During the past year in our project on the biosynthesis of Sec, we completed and published the entire pathway of Sec biosynthesis in eukaryotes and archaea. The roles of the two selenophosphate synthetases, SPS1 and SPS2, which were both previously thought to provide the active selenium donor in the biosynthesis of Sec, were further characterized. We found that SPS2 is indeed the enzyme that synthesizes monoselenophosphate, the active donor, while SPS1 has a different role that may not be involved in selenium metabolism. These studies resolved important questions in selenium biology on how selenium is inserted into Sec to be subsequently incorporated into protein. We have also shown in the last few years that the methyl group attached to the ribosyl moiety at position 34 on one of the two Sec tRNA isoforms (designated Um34) is responsible for the synthesis of stress-related selenoproteins (e.g., glutathione peroxidase 1), while the other isoform lacking this modification is involved in the synthesis of housekeeping selenoproteins (e.g., thioredoxin reductase 1). We are therefore focusing on the identification, isolation and characterization of the methylase responsible for Um34 addition to Sec tRNA. Bioinformatic analysis of methylases and potential methylases have provided us with several candidates that are currently being examined by knocking down their expression using siRNA technology. In addition, we previously reported that a factor designated SECp43 had a role in Um34 synthesis, and in the past year, we have generated the conditional knockout mouse of SECp43. This mouse is currently being characterized.